1. Field of the Invention
The industrial production of proteins coded by specific genes such as those encoding hormones, clotting factors, virus proteins, insulin, interferon and others, involves selecting and isolating gene sequences from viral, eukaryotic and other RNA or DNA, splicing of the sequences in the form of DNA into DNA vectors to provide recombinant DNA, and introducing said recombinant DNA into host cells capable of expressing these genes. The vectors may impart to the transformed host cells a phenotypic trait used for isolation and cloning purposes. The gene products are isolated from cell cultures by usual techniques.
Up to now, efforts in this field have usually been based on the adaptation of microorganisms as host cells for expressing genes of interest. Indeed, bacteria are often the organisms of choice since they can be grown rapidly, in large quantities and at low cost. Foreign DNA can be introduced easily into bacterial cells by using vectors such as plasmids, cosmids, viruses and the like.
Use of bacterial hosts, however, is not always sufficient to obtain expression of mature eukaryotic proteins. Many important eukaryotic proteins are modified by glycosylation, acetylation, phosphorylation, specific proteolytic cleavage and other forms of processing. In many instances post-transcriptional and post-translational modifications are crucial in determining the final biological properties of protein products. Non-proteolyticpost-translational modifications of certain proteins such as glycosylation, acetylation, phosphorylation and others may not occur correctly, if at all, in bacteria or cell types significantly different from the cell type in which the gene product of interest is normally produced in the organism. The correct form of these modifications may prove critical in the synthesis of fully competent gene products by molecular cloning techniques. For example, when the gene product is a glycoprotein, such as the haemagglutinin of influenza virus, the precise nature of glycosylation may influence the efficiency of antibodies raised against this synthetic protein to protect human beings or animals against influenza virus infections. Moreover, when glycosylation, acetylation or phosphorylation or other modifications are required to stabilize, activate, or mediate intracellular transport or excretion of a protein, the precision of these modifications may be critical to the utility of these genetically engineered protein products in practice.
Because of the above-mentioned shortcomings of using bacteria for the synthesis of complex gene products, there have been a number of attempts to introduce DNA encoding specific eukaryotic proteins into eukaryotic cells. DNA can be introduced by co-transformation into suitable mutant cells which are deficient in the production of a particular enzyme, such a thymidine kinase or hypoxanthine phosphoribosyl transferase. Then by culturing the cells in a selective medium deficient in the enzyme product, transformed cells may be selected for by their ability to grow, i.e., their ability to produce the enzyme. Alternatively, cells can be co-transfected in a positive sense to add a gene that will transduce cells to become selectively resistant to a drug or selective medium such methotrexate, neomycin, or others. Although many of these attempts have enjoyed some measure of success, in most cases the yield of mature gene products has been quite limited.
In order to maximize the yields of expression of fully competent gene products of interest, it would be desirable that the host expression unit system employed consists of an expression vector derived from one cell type or organism that is capable of synthesizing the said fully competent gene products, and that this expression vector directs the synthesis of said gene products in the same or similar cell type or organism as host.
2. Description of the Prior-Art
The heat-shock phenomenon has been studied most extensively in Drosophila melanogaster. For a review, see Ashburner and Bonner (1979) Cell 17: 241-254. When Drosophila cells or organs, normally at about 25.degree. C., are exposed to a heat treatment at 35.degree.-37.degree. C., a family of heat-shock genes is activated and most of the genes active at 25.degree. C. are no longer transcribed. Seven genes code for polypeptides with molecular weights between 22,000 and 84,000 daltons. During heat treatment these heat-shock polypeptides are synthesized almost exclusively and after 8 hrs, represent 10% of the total cellular protein [Arrigo, P. (1979) Ph. D. Thesis, University of Geneva]. During heat treatment of Drosphila cells, much of the polysome bound mRNA codes for heat-shock proteins [McKenzie et al. (1975) 72: 1117-1121; Mirault et al. (1978) Cold Spring Harbor Symp. Quant. Biol. 42: 819-827].
All seven Drosophila heat-shock protein genes have been cloned. See, Livak et al. (1978) PNAS 75: 5613-5617; Schedl et al. (1978) Cell 14: 921-929; Craig et al. (1979) Cell 16: 575-588; Holmgren et al. (1979) Cell 18: 1359-1370; Wadsworth et al. (1980) PNAS 77: 2134-2137; Corces et al. (1980) PNAS 77: 5390-5393; Voellmy et al. (1981) Cell 23: 261-270. A number of the genes have been sequenced. See Karch and Torok (1980) Nucleic Acid Res. 8: 3105-3123, and Ingola and Craig (1982) PNAS 79: 2360-2364.
All eukaryotic organisms appear to possess heat shock genes. See Kelly and Schlesinger (1978) Cell 15: 1277-1288. Many of the heat shock genes appear to be conserved throughout widely diverse species, and Drosophila heat shock genes have been shown to be transcribed in mouse cells [Corces et al. (1981) PNAS 78: 7038-7042], frog cells [Voellmy and Rungget (1982) PNAS 79: 1776-1780], and monkey cells [Pelham (1982) Cell 30: 517-528]. Fusion genes consisting of Drosophila heat-shock gene regions and Herpes Simplex virus thymidine kinase gene regions are also transcribed in these heterologous cell systems. No evidence has been presented which would suggest that the protein products of these genes are formed. Pelham, H. and Bienz, M. (1982) p. 43-48 in Heat Shock from Bacteria to Man. Ed. Schlesinger, Ashburner and Tissieres. Cold Spring Harbour Press. Corces et al. (1982) p. 27-34 in Heat Shock from Bacteria to Man. Ed. Schlesinger, Ashburner and Tissieres. Cold Spring Harbour Press.